The objective of this proposal is to isolate and characterize the site of mineral deposition in matrix vesicles. This will be achieved using matrix vesicles, induced-vesicles and plasma membranes isolated from epiphyseal plate chondrocytes and from chondrocytes maintained in culture. Vesicles and membranes will be fragmented by physical and chemical disruptive techniques. For some experiments, recombinant vesicles will be formed by fusing membrane fragments into liposomes. Mineralization-competent membrane fragments and recombinant vesicles will be selected by calcium-loading and density gradient separation procedures. We will then determine the mineralization potential of vesicles and fragments by calcium binding and turbidity measurements. Chemical and morphological analyses will be used to characterize the lipid, protein and mineral components of the mineralizing fragments. By modifying the lipid composition of the recombinant vesicles we will examine the role of specific phospholipids in matrix vesicle mineralization. Finally, we propose to study the role of alkaline phosphatase in mineralization by preparing alkaline phosphatase-containing liposomes and measuring their mineralization potential. These liposomes will be used in recombination experiments to investigate the interaction of the enzyme with other membrane components.